1. Technical Field
The subject matter of the present invention relates to 5,10-methylene-tetrahydrofolate (CH.sub.2 FH.sub.4), therapeutic uses of this compound and compositions thereof. CH.sub.2 FH.sub.4 strongly modulates the in vivo antitumor effects of 5-Fluorouracil. Furthermore, the present invention additionally relates to a solution product isomer of CH.sub.2 FH.sub.4, tetrahydrofolate (FH.sub.4), which also strongly modulates the in vivo antitumor effects of 5-Fluorouracil. CH.sub.2 FH.sub.4 and FH.sub.4 both exist in a natural ((6R)-CH.sub.2 FH.sub.4 and (6S)-FH.sub.4) and unnatural ((6S)-CH.sub.2 FH.sub.4 and (6R)-FH.sub.4) diastereomeric form and both forms may be utilized for purposes of the present invention. In solution, CH.sub.2 FH.sub.4 and FH.sub.4 exist in chemical equilibrium, with requirements for millimolar formaldehyde concentrations to favor the balance toward CH.sub.2 FH.sub.4.
2. Background Information
The compound 5-Fluorouracil (5-FU) is possibly the most widely used anticancer drug in the world. In the 1970s and early 1980s, the prevailing opinion among cancer researchers was that the key biochemical lesion caused by 5-FU in tumor cells resulted from the drug's incorporation into RNA (Kufe et al., J. Biol. Chem. 256:9802 (1981) and Glazer et al., Mol. Pharmacol. 21:468 (1982)).
In 1982, using a specifically designed assay of the DNA enzyme, thymidylate synthase (TS) (EC 2.1.1.45), the present inventors established that the therapeutic mechanism of 5-FU against murine colon cancer was complete inhibition of TS or abrogation of TS activity (Spears et al., Cancer Res. 42:450-56 (1982)). In fact, the present inventors were the first to report a clinical correlation between TS level in a patient's cancer after 5-FU treatment and response (Spears et al., Cancer Res. 44:4144-50 (1984)). The finding has been confirmed by several research groups.
TS is the only intracellular source of new ("de novo") thymine synthesis, as the enzyme which catalyzes the methylation of deoxyuridylate to form thymidylate (thymine-2'-deoxyribose-5'-phosphate). Thymine is one of the four main building blocks of DNA, and its occurrence in DNA (vs. its absence in RNA) is the major structural difference between DNA and RNA. Thus, the activity of TS to make new thymidylate and DNA is essential to cell division, tissue regeneration and turnover, and tumor growth. The source of the methyl one-carbon group for synthesis of thymidylate is CH.sub.2 FH.sub.4 and its polyglutamates. The mechanism of methyl transfer by TS has recently been reviewed (K. T. Douglas, Medicinal Res. Rev. 7:441-75 (1987)). After initial weak binding of deoxyuridylate to TS, the enzyme catalyzes ring-opening of CH.sub.2 FH.sub.4 at the imidazole C11 ring. This may be the rate limiting step overall. The relative stability of tetrahydrofolate within the ternary complex, toward oxidation, suggests that the ring-opening occurs with the substitution at N5, in accordance with formation of an N5-iminium cation species (S. J. Benkovic, Ann. Rev. Biochem., 49:227- 51 (1980)). Covalent bonding between the methylene group and the C5-position of deoxyuridylate is accompanied by rapid hydride transfer from the C6-position of the ring-opened CH.sub.2 FH.sub.4 so that CH3- is formed on the C5 position of the nucleotide. This leads rapidly to expulsion of the two products from the TS binding site(s), i.e., thymidylate and dihydrofolate. TS is the only enzyme which oxidizes reduced folates to dihydrofolate, which is then converted back to tetrahydrofolate by another enzyme, dihydrofolate reductase. In general, the limiting intracellular factors in this biochemical pathway for making thymine are, in order of increasing scarcity, deoxyuridylate, dihydrofolate reductase, TS, and then CH.sub.2 FH.sub.4. Thus, a decrease in thymidine production through the TS pathway can result from nutritional deficiencies which decrease CH.sub.2 FH.sub.4 production (i.e., primary folate deficiency, B12, B6, and other B-vitamin deficiencies which impair folate one-carbon metabolism), or from antimetabolites drugs such as 5-FU or methotrexate. Methotrexate inhibits dihydrofolate reductase, thus blocking the regeneration of tetrahydrofolates from dihydrofolate. 5-FU and other fluorinated pyrimidines (for example, floxuridine, FUDR or trifluoromethylthymidine) block TS activity through formation of the specific metabolite for this effect, fluorodeoxyuridylate (FdUMP), discussed below.
Inhibition of TS activity leads to "thymineless cell death" or "unbalanced cell growth," whereby RNA and protein synthesis, and cell enlargement, occur in the absence of adequate new DNA synthesis (see Goulian et al., Adv. Exp. Med. Biol. 195:89-95 (1986), and refs. therein). In blood cells, such unbalanced cell growth can lead to megaloblastic anemia, macrocytosis, and bone marrow failure.
The mechanism of inhibition of TS by FdUMP has been studied intensively for the past two decades (see Santi et al., Biochem., pp. 8606-13, (1987) and refs. therein). In the absence of CH.sub.2 FH.sub.4, FdUMP binds TS extremely weakly. However, in the presence of a large excess of CH.sub.2 FH.sub.4, even low levels of FdUMP will bind tightly to TS, by forming inhibitory TS-FdUMP-CH.sub.2 FH.sub.4 ternary complexes. In the presence of excess CH.sub.2 FH.sub.4, such ternary complexes are stable and no significant TS activity occurs. The molecular basis for the ternary complex is that after CH.sub.2 FH.sub.4 ring-opening to form a covalent bond to FdUMP in the TS enzyme pocket (analogous to the normal reaction with deoxyuridylate), no hydride ion transfer can occur. Thus, no dihydrofolate is formed and the covalently-bonded FdUMP-CH.sub.2 FH.sub.4 only leaves the enzyme site with great difficulty, as long as free CH.sub.2 FH.sub.4 is present in substantial excess. If the CH.sub.2 FH.sub.4 concentration is relatively low, the ternary complex dissociates back to starting products, including free, active TS.
Thus, TS inhibition can occur with only trace amounts of FdUMP in slight excess over TS molecules; however, a specific condition must occur in that 5-10-methylenetetrahydrofolate (CH.sub.2 FH.sub.4) or tetrahydrofolate (FH.sub.4) (and their polyglutamates) must be present in high concentration. Stated more simply, CH.sub.2 FH.sub.4 is like a "glue" that holds the FdUMP onto the TS enzyme and therefore inhibits TS activity. However, CH.sub.2 FH.sub.4 is also a powerful growth factor, for promotion of purine, protein, and lipid metabolism, as well as pyrimidine synthesis; thus, CH.sub.2 FH.sub.4 administration for the purpose of promotion of TS inhibition by FdUMP may be expected to also increase the degree of "unbalanced cell growth."
CH.sub.2 FH.sub.4 is a normal intracellular metabolite of the B-vitamin, folic acid, for use in thymidylate synthesis by TS. The same is true with respect to the polyglutamates of CH.sub.2 FH.sub.4. However, CH.sub.2 FH.sub.4 is also used by several other enzymes including CH.sub.2 FH.sub.4 reductase (EC 1.1.99.15), serine hydroxymethylase (EC 2.1.2.1), and Cl-tetrahydrofolate synthase and CH.sub.2 FH.sub.4 dehydrogenase (EC 1.5.1.5). These interconversions using CH.sub.2 FH.sub.4 are essential for purine synthesis, amino acid synthesis (i.e., serine and methionine), and lipid metabolism through the re-methylation of methionine. Thus, CH.sub.2 FH.sub.4 is located at a metabolic branch point as a substrate for at least 4 different enzymes (Green et al., Biochem. 27:8014-22, (1988), S. J. Benkovic, Ann. Rev. Biochem. 49:227-51 (1980) and Schirch et al., Arch. Biochem. Biophys. 269:317-80 (1989)). This explains the fact that intracellular CH.sub.2 FH.sub.4 is normally present in low concentrations, below 1.0 micromolar. Recent measurements have shown that intracellular CH.sub.2 FH.sub.4 levels are typically low, and virtually always lower than tetrahydrofolate, using the bacterial L. Casei TS-[3H]FdUMP ligand binding assay (Priest et al., Cancer Res. 48:3398-3404 (1988), and refs. therein). The present inventors have modified this assay (Adv. Exp. Med. Biol. 244:98-104 (1988) and Invest. New Drugs 7:27-36 (1989)) and reported relatively low levels of CH.sub.2 FH.sub.4 (much below 1.0 micromolar) in patients' cancer biopsy specimens despite administration of high doses of leucovorin (LV) (Proc. Am. Soc. Clin. Oncol. 8:69 (1989)). Furthermore, these observations of the present inventors led to administration of the amino acid, L-serine, to patients in an attempt to convert the tetrahydrofolates (in various polyglutamate forms, present in large excess) to CH.sub.2 FH.sub.4 (and polyglutamates). These results have suggested that increased FH.sub.4, rather than CH.sub.2 FH.sub.4, may be therapeutic. The inventors have recently published the only comparative data that exist for the different major intracellular one-carbon forms of folates (Biochem. Pharmacol. 38:2985-93 (1989)), showing that of all of these, CH.sub.2 FH.sub.4 (at least, as the monoglutamate) is the best folate form for formation of TS-FdUMP-folate ternary complexes, and that a concentration of CH.sub.2 FH.sub.4 in excess of 1.0 micromolar is desirable for this effect. CH.sub.2 FH.sub.4 was found to be four times stronger than the next best folate, tetrahydrofolate, and about 100 times stronger than LV. However, to maintain CH.sub.2 FH.sub.4 as this form (vs. aqueous dissociation to FH.sub.4), formaldeyde was required to be present in great excess over the folate at these micromolar concentrations of folate.
Leucovorin (referred to as LV, or folinic acid) is (6R,S)-5-formyl-tetrahydrofolate and has been available commercially for decades for the treatment of folic acid (the B-vitamin) deficiency states (The Pharmacologic Basis of Therapeutics, 4th ed. (Goodman et al., eds.) The MacMillan Co., Toronto, pp. 1431-44 (1970)). In 1982, the first clinical reports of the usefulness of LV as a modulator of 5-FU in cancer treatment appeared. (Machover et al., Cancer Treat. Rep. 66:1803-07 (1982)). LV addition to 5-FU appeared to approximately double response rates in patients with gastrointestinal cancers. This result was confirmed in several subsequent studies. (For an extensive review, see Grem et al., Cancer Treat. Rep. 71:1249-64 (1987)). Currently, LV addition to 5-FU therapy is community standard practice in the United States.
The mechanism of leucovorin (LV or folinic acid) improvement in the antitumor therapy of 5-FU and floxuridine (FUDR) has been shown in several studies to be due to improved TS inhibition associated with increased intracellular (6R)-CH.sub.2 FH.sub.4 and (6S)-tetrahydrofolates. However, LV appears to be only partially effective in the goal of promoting complete TS inhibition by FdUMP in vivo. For an in vitro example, researchers have shown that TS inhibition after 5-FU, while improved by LV, was still clearly incomplete (Keyomarsi et al., J. Biol. Chem. 263:14402-09 (1988)). In part, this may have been related to saturation of obtainable summed pools of CH.sub.2 FH.sub.4 +FH.sub.4 at about a 5-fold increase over baseline at 30 hr LV exposure. Thus, maximum synergy of LV was obtained at less than 1.0 micromolar exposure, with no further improvement at higher concentrations although human plasma folates (LV and methyltetrahydrofolate, MTHF) are higher than this after high-dose LV administration (Doroshow et al., NCI Monogr, 5:171-74 (1987)). A related observation may be that addition of high-dose folic acid (140 mg/m.sup.2) to 5-FU therapy appears to be associated with an increase in toxicity without improved response rates (Asbury et al, Am. J. Clin. Oncol. 10:47-49 (1987)).
In fact, decreasing synergy has been shown for LV addition to FUDR at concentrations above 0.5 micromolar, when the colon cancer cells were previously folate-deficient (Davis et al., Mol. Pharmacol. 35:422-27 (1989)). Also, others have shown in vivo in mice that expansion of breast tumor CH.sub.2 FH.sub.4 pools was a maximum of less than two-fold over baseline despite massive LV dosing (180 mg/kg.times.8 over 48 hr) (Wright et al., Cancer Res. 49:2592-96 (1989)). These observations are mirrored in recent clinical trials comparing the therapeutic outcome in colon cancer, in which low-dose LV (20 mg per square meter) was more effective than high-dose LV (200 mg per square meter) in terms of both tumor response rate and patient survival (Poon et al., J. Clin. Oncol. 7:1407-18 (1989)). The lack of effectiveness of high-dose LV in promoting complete TS inhibition was suggested by researchers based on tumor biopsy analyses in breast cancer patients: LV increased TS inhibition from an average of 30.+-.13 to 71.+-.14%, with responding patients showing the higher percentages of TS inhibition than non-responders (Swain et al., (J. Clin. Oncol. 7:890-99 (1989)).
In view of the above, the present inventors realized the potential of the direct administration of CH.sub.2 FH.sub.4 to patients receiving 5-FU, as such a course of action would maximize TS inhibition.
The desirability and ability to use CH.sub.2 FH.sub.4 in the method of the present invention have never been obvious for various reasons.
For example, CH.sub.2 FH.sub.4 as a compound in solution has enjoyed a general reputation of being extremely unstable. (Temple et al., "Chemical and Physical Properties of Folic Acid and Reduced Derivatives," In Folates and Pterins (Blakely et al., eds.), Vol. 1, pp. 61-63 (1984) and Wright et al., Cancer Res. 49:2592-96 (1989)). In solution, it is generally known to exist in equilibrium with FH.sub.4, requiring excess formaldehyde (CH.sub.2 O) to favor the equilibrium toward CH.sub.2 FH.sub.4.
Under anaerobic conditions, such as made possible for clinical administration of CH.sub.2 FH.sub.4 or FH.sub.4 by a closed, delivery system (U.S. Pat. No. 4,564,054), powdered FH.sub.4 is stable even at room temperature, for a year or more (Caldwell et al., Prep. Biochem. 3:323-26 (1973)).
Additionally, published data on the clinical tissue levels of CH.sub.2 FH.sub.4 and FH.sub.4 in patients have been limited, and it is well known that LV can be given in gram-size doses (Grem, et al., supra.). LV is an extremely powerful folate (B-vitamin) that is one-hundred times stronger than folic acid in correcting nutritional folate deficiency. As little as 1.0 mg of LV will correct folate deficiency as a single dose (The Pharmacological Basis of Therapeutics, supra.). Thus, it is logical to assume that tumor CH.sub.2 FH.sub.4 and FH.sub.4 levels might reach saturation levels from high dose LV.
Finally, it appears that no published studies exist on the toxicological aspects of CH.sub.2 FH.sub.4 or FH.sub.4. More specifically, there seems to be no available published work on either in vitro or in vivo effects of direct exposure of living cells to CH.sub.2 FH.sub.4 or FH.sub.4 except to rescue methotrexate toxicity (Kisliuk et al., Cancer Treat. Rep. 61:647-50 (1977)).
Thus, in view of the structural properties of CH.sub.2 FH.sub.4 and FH.sub.4 as well as the lack of information regarding the biological effects of CH.sub.2 FH.sub.4 and FH.sub.4, the present invention is quite remarkable. CH.sub.2 FH.sub.4 is utilized to potentiate or modulate the antitumor effects of the chemotherapeutic agent 5-FU.
L. R. Hughes (Eur. Pat. Appl. EP 284,3380 and Chem. Abstr. 110:95789 (1989)) has described a novel folate analog as a TS inhibitor and antitumor agent. However, the discovery is clearly radically different from the present invention. The analog does not occur naturally, is absent two nitrogen atoms, is not reduced, and has a reactive propargyl group attached to the glutamate moiety. Also, no mention is made of 5-FU.
Interleukin-2 has been proposed as a modulator of tetrahydrobiopterin (U.S. Pat. No. 4,752,573); however, interleukin-2 is an oligopeptide having no resemblance to leucovorin, and no claim for TS inhibition or interaction with 5-FU is made.
A patent for radiolabeled assay of folates (U.S. Pat. No. 4,136,159) has no therapeutic pharmaceutical intent, and makes no mention of TS inhibition.
Various patents exist for other, unnatural folate analogs, including quinazolines and dideazatetrahydrofolates as inhibitors of enzymes such as folylpolyglutamyl synthetase (e.g., see Chem. Abstr. 110: P39366p (1989)). However, these are unnatural analogs which have distinct chemical, structural differences from CH.sub.2 FH.sub.4.
The European patent application (EP 266,042) of Wood et al. describes a process for separation of diastereomers of LV, as well as (6R)- and (6S)-tetrahydrofolates. No use of CH.sub.2 FH.sub.4 or FH.sub.4 as a potentiator of TS inhibition by FdUMP (and thus 5-FU and other fluoropyrimidines) is claimed in the document.
All U.S. patents and publications referred to herein are hereby incorporated by reference.